The Relative Effect of Sterols and Hopanoids on Lipid Bilayers: When

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The Relative Effect of Sterols and Hopanoids on Lipid Bilayers: When Comparable Is Not Identical David Poger, and Alan Edward Mark J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 20 Nov 2013 Downloaded from http://pubs.acs.org on November 30, 2013

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The Journal of Physical Chemistry

The Relative Effect of Sterols and Hopanoids on Lipid Bilayers: When Comparable Is Not Identical David Poger∗,† and Alan E. Mark†,‡ The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane QLD 4072, Australia, and The University of Queensland, Institute for Molecular Bioscience, Brisbane QLD 4072, Australia E-mail: [email protected]

Phone: +61 (0)7 3365 7562. Fax: +61 (0)7 3365 3872 KEYWORDS: Membranes, Sterols, Hopanoids, Diploptene, Bacteriohopanetetrol, Ordering

∗ To

whom correspondence should be addressed of Chemistry and Molecular Biosciences, The University of Queensland ‡ Institute for Molecular Bioscience, The University of Queensland

† School

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Abstract Sterols are the hallmarks of eukaryotic membranes where they are often found in specialised functional microdomains of the plasma membrane called lipid rafts. Despite some notable exceptions, prokaryotes lack sterols. However, growing evidence has suggested the existence of raft-like domains in the plasma membrane of bacteria. A structurally related family of triterpenoids found in some bacteria called hopanoids, has long been assumed to be bacterial surrogates for sterols in membranes. Although the effect of sterols, in particular cholesterol, on lipid bilayers has been extensively characterised through experimental and simulation studies, those of hopanoids have hardly been investigated. In this study, molecular dynamics simulations are used to examine the effect of two hopanoids, diploptene (hop-22(29)-ene) and bacteriohopanetetrol ((32R,33S,34S)-bacteriohopane-32,33,34,35-tetrol), on a model bilayer. The results are compared with those obtained for cholesterol and a pure phosphatidylcholine bilayer. It is shown that diploptene and bacteriohopanetetrol behave very differently under the conditions simulated. Whereas bacteriohopanetetrol adopted a cholesterol-like upright orientation in the bilayer, diploptene partitioned between the two leaflets inside the bilayer. Analysis of various structural properties (area per lipid, electron density profile, tilt angle of the lipids, conformation and order parameters of the phosphatidylcholine tails) in bacteriohopanetetroland cholesterol-containing bilayers indicates that the condensing and ordering effect of bacteriohopanetetrol is weaker than that of cholesterol. The simulations suggest that the chemical diversity of hopanoids may lead to a broader range of functional roles in bacterial membranes than sterols in eukaryotic membranes.

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Introduction Lipids constitute an extraordinary diverse class of biomolecules including glycerophospholipids, sphingolipids and sterols. They mostly reside in cellular membranes and the eukaryotic plasma membrane alone can contain hundreds of different lipid species. Strikingly, sterols can represent up to 20% of all lipids in plant membranes and 30–50% in animal membranes. Animal and yeast membranes contain only one type of sterol, cholesterol (depicted in Figure 1A) and ergosterol, respectively, while plant membranes incorporate a variety of sterol species. Sterols play a critical role in the formation of microdomains in the plasma membrane called lipid rafts which in turn are involved in membrane trafficking and signal transduction. 1 Lipid rafts are compartmentalised functional platforms with physicochemical properties that are distinct from the bulk membrane. In particular, sterols can modulate the ordering of lipid chains, 2,3 condense the packing of lipids, 4 restrict the lateral diffusion of lipids and proteins, 5,6 regulate the function of proteins 7 and alter the thermotropic phase transition between the solid-ordered (gel Lβ and Lβ " ) and liquid-disordered (fluid Lα ) phases of lipid bilayers. 8 Specifically, sterols can induce an intermediate lamellar phase referred to as the liquid-ordered phase (Lo ). The biosynthesis of sterols from squalene is almost ubiquitous in eukaryotes but, except for a few notable exceptions including some Proteobacteria (Methylococcus capsulatus, 9 Methylosphaera hansonii, 10 Methylobacterium organophilum, 11 Stigmatella aurantiaca 12 and Nannocystis exedens 13 ) and a Planctomycete (Gemmata obscuriglobus 14), it is virtually non-existent in prokaryotes. Nonetheless, even bacteria that do not produce sterols are not devoid of triterpenoids. A large number of bacteria contain pentacyclic triterpenoids. Some are based on a hopane skeleton and are called hopanoids. 15 As can be seen in Figure 1, hopanoids (for example, diploptene in Figure 1B and bacteriohopanetetrol in Figure 1C) are structurally related to sterols (such as cholesterol in Figure 1A). The main difference is that the polar hydroxyl headgroup in sterols is located in the A ring of the steroid nucleus (on carbon 3) whereas it is in the E ring of hopanoids (on carbon 21). Also, unlike sterols which always have a polar hydroxyl headgroup, hopanoids display considerable diversity in the size and chemical nature of the headgroup. Three families of hopanoids have been 3



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identified: C30 derivatives of hopane itself (for example diploptene and diplopterol), C35 derivatives that are based on a 29-pentylhopane skeleton (bacteriohopane) which can be heavily functionalised (for example bacteriohopanetetrol and aminobacteriohopanetriol), and bacteriohopane derivatives which commonly carry extra methyl groups on the fused ring system (the “backbone”). Given their structural similarity to sterols, hopanoids have long been assumed to be functional analogues to sterols in bacteria. 16 Surprisingly however, only a few studies have examined their properties in detail. It has been shown that diplopterol 17 and aminobacteriohopanetriol 18 could enhance the ordering of lipid chains and modulate phase transitions in membranes. Condensation of phosphatidylcholine monolayers has also been observed when mixed with bacteriohopanetetrol and a glycoside derivative. 19–21 Hopanoids may also confer additional properties on membranes such as resistance to low and high pH, 22,23 high temperature, 24 high ethanol concentration, 25 desiccation 26 and oxidative stress. 27,28 Interestingly, structurally and functionally specialised microdomains in the plasma membrane of Bacillus subtilis 29 and Gloeobacter violaceus 30 have been reported. A detergent-resistant membrane fraction extracted from Crocosphaera watsonii was also shown to be enriched in hopanoid. 31 Consistent with the hypothesis of being bacterial sterol surrogates, hopanoids have therefore been implicated as a putative central component in such bacterial lipid rafts. 31 Nonetheless, despite their abundance, little is known regarding the behaviour of hopanoids in biological membranes at a molecular level. The aim of this study was to investigate i. whether hopanoids can truly be regarded as bacterial analogues to sterols, and, ii. to what extent prokaryotic hopanoid-enriched membrane microdomains may be compared with eukaryotic sterol-enriched rafts. Using molecular dynamics simulation, we have examined the membrane partitioning, ordering and condensing properties of two model hopanoids: diploptene and bacteriohopanetetrol. Diplotene, a C30 hopane derivative, has a terminal double bond as a headgroup (Figure 1B) and is widespread in hopanoid-producing bacteria but generally in small amounts 32,33 . Bacteriohopanetetrol (BHT) is a polyhydroxylated C35 hopanoid (Figure 1C) and one the most abundant hopanoids in membranes. 19,25,27 The effect of diploptene and BHT is compared with that of cholesterol in a model “raft-like” lipid bilayer. It is shown that

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diplotene and BHT exhibit dramatically different behaviours in a lipid bilayer under the conditions simulated. Furthermore, it is shown that the ordering and condensing effect of the hopanoids examined on a lipid bilayer is weaker than that of cholesterol. The simulations suggest that the chemical diversity of hopanoids may lead to distinct functions in cellular membranes.

Methods Model systems Seven different systems were examined. The first consisted of a pure 128-POPC bilayer taken from a previous study. 34 Four other systems consisted of a hydrated, preassembled mixed bilayer containing equimolar amounts of POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine) and cholesterol, bacteriohopanetetrol ((32R, 33S,34S)-bacteriohopane-32,33,34,35-tetrol) or diplotene (hop-22(29)-ene). For diploptene, a different lipid composition with a POPC-to-diploptene ratio of 10:1 was also simulated. All bilayers contained 576 lipid molecules in total. The starting orientations of cholesterol and hopanoids in the bilayer were chosen with their long axis aligned with the normal of the plane of the bilayer (taken as the z-axis). In the case of cholesterol and bacteriohopanetetrol (BHT), the hydroxyl groups faced outwards; that is they were directed towards the interface with water, the usual orientation for cholesterol. In the case of diploptene, two initial orientations were examined: all the isopropenyl groups facing either the core of the bilayer ( “inward” orientation as shown in Figure 2A) or water (“outward” orientation, as in Figure 2B). The pure POPC bilayer was hydrated by 5941 water molecules (about 46 waters per lipid). In the case of the hopanoid- and cholesterol-containing bilayers, the hydration was approximately 27 water molecules per lipid or in total 15455 water molecules to ensure a fully hydrated state. 35 The leaflets of each bilayer were constructed by replicating a pair of lipids (POPC–cholesterol or POPC–hopanoid) separated by about 0.71 nm on a 12 × 12 grid, allowing an initial area per lipid of about 0.50 nm2 for all lipid molecules.

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Simulation parameters All simulations were performed using the G ROMACS package, version 3.3.3 36 in conjunction with the G ROMOS 54 A 7 united-atom force field. 37 Parameters for cholesterol were obtained from the Automated Topology Builder (ATB). 38 The hopane backbone was based on a cyclopenta[a]chrysene skeleton (referred to as the hopanoid nucleus from here on). The parameters for the hopanoid nucleus were derived from those of the steroid nucleus in cholesterol. The atomic partial charges in the 5,6,7,8-tetrahydroxyoctan-2-yl headgroup in bacteriohopanetetrol and the isopropenyl headgroup in diploptene were derived from those calculated for model molecules using the ATB, namely (2S,3R,4R)-heptane-1,2,3,4-tetrol and prop-1-en-2-ylcyclopentane, respectively. Each system was simulated under periodic boundary conditions in a rectangular box. The length of all covalent bonds within the lipids were constrained using the L INCS algorithm. 39 The geometry of the Simple Point Charge (SPC) water molecules 40 was constrained using S ETTLE. 41 The temperature of the system was maintained by coupling to an external temperature bath at the reference temperature of 298 K with a coupling constant of 0.1 ps using a Berendsen thermostat. 42 This temperature is above the gel-to-fluid phase-transition temperature of a pure POPC bilayer (270.6 K). 43 The pressure was kept at 1 bar in the lateral and normal directions with respect to the bilayer by weakly coupling to an anisotropic pressure bath 42 using an isothermal compressibility of 4.6 × 10−5 bar−1 and a coupling constant of 1 ps. A 2-fs timestep was used. A twin-range cutoff scheme was used for the evaluation of non-bonded interactions: interactions falling within the 0.8-nm short-range cutoff were calculated every step whereas interactions falling within the 1.4-nm long-range cutoff were updated every 10 fs, together with the pair list. A reaction-field correction 44 was applied to account for the truncation of the electrostatic interactions beyond the long-range cutoff using a relative dielectric permittivity constant of 62, as appropriate for SPC water. 45 The mixed-bilayer systems (that is the POPC/cholesterol, POPC/BHT and POPC/diploptene bilayers) were each simulated four times starting from different initial velocities, whereas the pure POPC bilayer was simulated three times. Each system was first energy-minimised and then sim6


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ulated at 50 K for 10 ps. The temperature was then increased gradually over 150 ps until the final simulation temperature was reached. The equilibration of the systems was monitored by examining the time evolution of the potential energy and the area per lipid of the system. Once the systems were equilibrated, data were collected for 200 ns for the simulations. The simulations of a pure POPC bilayer were taken from an earlier work 34 and extended in order to obtain a production time of 200 ns similar to the other systems. An overview of the simulations performed is given in Table 1.

Analysis Area per Lipid The area per lipid AL was calculated using a grid-based approach. A grid was generated parallel to the plane of the bilayer (i.e. aligned with the xy-plane) and centred at the centre of mass of the bilayer. The grid spacing was set to 0.01 nm. The centre of mass of each lipid molecule (or subset of atoms) was projected on to the grid. The area of each grid cell (10−4 nm2 ) was assigned to the nearest lipid, taking the periodic boundary conditions into account. Electron Density and Bilayer Thickness The profiles were computed by dividing the simulation box into 150 slices along the bilayer normal (z-axis). The partial density for each atom in each slice was weighted by the number of electrons and averaged over the whole simulation. The bilayer thickness DHH was defined as the distance between the maxima of the electron density of the whole system. Tilt Angle of Lipid Molecules The orientation of the sn-1 palmitoyl and sn-2 oleoyl tails in POPC was described using the tilt angles γ1 and γ2 , respectively, of the vector joining the first and the last carbon atoms of each acyl chain with respect to the bilayer normal projected along the z-axis away from the plane

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of the membrane. For cholesterol and biohopanoids, the vector used for the tilt angle γN was the first principal axis of the steroid and hopanoid nuclei, respectively. The steroid nucleus consists of the cyclopenta[a]phenanthrene tetracyclic moiety corresponding to gonane in cholesterol (carbons 1–17 in cholesterol). By analogy the hopanoid nucleus was taken as the atoms in the cyclopenta[a]chrysene-derived pentacyclic core of hopane or 13a,17-propano-13(17)a-homogonane (carbons 1–21 in hopanoids). Conformation of the Palmitoyl Chains in POPC The degree of gauche–trans isomerisation in the sn-1 palmitoyl tail of POPC was estimated based on two properties: the total number of gauche rotamers per chain (ng ) and the occurrence of successive trans rotamers per chain. A sequence consisting of n consecutive trans conformers is denoted tn. Note the sequence t13 corresponds to an all-trans conformation. In the simulations, the torsion angles φ in the palmitoyl chains were classified as gauche when |φ | ∈]30°; 90°] and trans when |φ | ∈]150°; 180°]. 46 The occurrence p(ti ) of a sequence ti is defined as the ratio between the number of sequences ti and the total number of sequences tn,n∈[1;13] calculated throughout the simulation in the sn-1 palmitoyl tail. Order Parameter of Covalent Bonds The order parameter SCD of a carbon–deuterium bond measures the relative orientation of the C–D bonds with respect to the bilayer normal (taken as the z-axis). The order parameter SCD of a bond is defined as: 1 SCD = &3 cos2 β − 1' 2

(1)

where β is the angle between a C–D bond and the normal to the bilayer. The angular brackets denote an ensemble average over all the lipids and the simulation. As G ROMOS 54 A 7 is an unitedatom force field wherein aliphatic hydrogens are treated implicitly and incorporated into the carbon to which they are bound, the position of the deuteron was constructed based on the positions of the neighbouring heavy atoms (carbons) assuming sp3 geometry for tetrahedral carbons and sp2 8


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geometry for alkenic carbons.

Results Orientation of Diploptene and Bacteriohopanetetrol in a Lipid Bilayer Figure 2 presents snapshots of the structure of the POPC bilayer mixed with diplotene (panels A and B) or bacteriohopanetetrol (panel C) in a 1:1 ratio at the beginning (upper half of the panels) and the end of simulations (lower half of the panels). Panels A and B show the initial structure of the bilayer with the diploptene molecules in an inward and outward orientation, respectively. It is evident that after 300 ns of simulation, almost all the diploptene molecules migrated to the centre of the bilayer between the two POPC leaflets, regardless of the initial orientation of the diplotene molecules. In contrast, as displayed in panel C, bacteriohopanetetrol (BHT) maintained an upright orientation in the POPC bilayer throughout all the simulations with the polyhydroxylated headgroup facing water in a cholesterol-like fashion. The rearrangement of the diploptene molecules was further quantified by calculating the average tilt angle &γD ' of the hopanoid nucleus in each leaflet with respect to the normal of the bilayer (z-axis) (Figure 3A) and the average distance &zD ' between the centre of mass of the diplotene molecules and the centre of the bilayer (Figure 3B). Note, &γD ' and &zD ' were computed and averaged over all the diploptene molecules within the same leaflet for each configuration of the simulation. The variation of &γD ' and &zD ' clearly indicate that the reorientation of diplotene began in the very early stages of the simulations. The partitioning in between the two leaflets of the lipid bilayer was concomitant with the rotation of the molecules. Within 200 ns of simulation, the average tilt angle &γD ' of the hopanoid nucleus had reached equilibrium at about 51°. BHT in contrast maintained a stable upright orientation with an average tilt angle &γB ' of 14° (inset in Figure 3A). Diplotene exhibited the same behaviour in the simulation of POPC/diploptene bilayers which contained 9 mol% diploptene in an inward or outward initial orientation (data not shown).

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Relative Condensing Effect of Bacteriohopanetetrol and Cholesterol The relative condensing effect of bacteriohopanetetrol on the structure of the bilayer with respect to that of cholesterol is examined through different properties, in particular the area per lipid, the bilayer thickness, the tilts of the lipid molecules and the conformation of the acyl chains. Area per Lipid The area per lipid AL was calculated for each species, namely POPC, cholesterol and BHT. Figure 4 shows the average probability distribution of AL for POPC (Figure 4A) and cholesterol and bacteriohopanetetrol (Figure 4B) in the relevant simulations. In each case, AL was estimated using the position of the centre of mass of the entire lipid molecule and the headgroup only. The latter consisted of the atoms in the choline moiety in POPC, the hydroxyl group in cholesterol and the 5,6,7,8-tetrahydroxyoctan-2-yl group in BHT. The distributions of AL obtained for pure POPC with the two methods overlap exactly. In contrast, in the mixed systems, the AL values for POPC, cholesterol and BHT vary substantially depending on the methodology employed. Specifically, the position, the width and the intensity of the peaks change. When using the centre of mass of the headgroup to calculate AL , the spread of the values is greater, leading to broader (50% on average) and less intense (30% on average) peaks compared to that obtained when taking the centre of mass of the whole lipid. This demonstrates the intrinsic difficulty in assigning the area per lipid in mixed systems. However, the trend in the variation of AL in the simulations of POPC/cholesterol and POPC/BHT bilayers is consistent. In the presence of cholesterol and BHT, the probability distribution of AL for POPC is moved by approximately 0.15 nm2 and 0.20 nm2 , respectively, towards lower AL values, compared to that in a pure POPC bilayer, indicating that the two molecules enhance tighter packing of the lipids than in a fluid phase, with cholesterol having the largest condensing effect. The peaks calculated for the distribution of AL for POPC and cholesterol in the POPC/cholesterol bilayer are also sharper than those for POPC and BHT in the POPC/BHT bilayer which also suggests that lipids are more ordered in the presence of cholesterol. The average values calculated for AL are listed in Table 2. As shown previously, 34 10


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the value of &AL ' (0.626 nm2 ) computed for a pure POPC bilayer is in good agreement with the experimental values measured for a fluid bilayer. It is decreased in the presence of BHT (0.459– 0.473 nm2 ) and cholesterol (0.414–0.418 nm2 ). Note, there is a variation of less than 1% and 3% between the two methods for the evaluation of AL in the POPC/cholesterol and POPC/BHT bilayers, respectively. The values of AL calculated for the POPC/cholesterol bilayer lie in the range of the area per lipid estimated from experiment, in particular from the studies of equimolar phosphatidylcholine–cholesterol mixed bilayers by Ipsen et al. 47 (AL of 0.42 nm2 and 0.32 nm2 for DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and cholesterol, respectively) and Pan et al. 48 (AL of 0.41 nm2 and 0.37 nm2 for DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and cholesterol, respectively; and 0.49 nm2 and 0.29 nm2 for DOPC (1,2-dioleoyl-sn-glycero-3phosphocholine) and cholesterol, respectively). To our knowledge, no experimental value for AL has been published for phosphatidylcholine–hopanoid mixtures. Electron Density Profiles The electron density profiles across the lipid bilayer in the POPC, POPC/cholesterol and POPC/BHT bilayers are displayed in Figure 5. The total electron density profiles of the hydrated bilayers are shown in panel A. The contributions from different components of the systems are displayed in panel B—namely: choline (Cho), phosphate (P) and glycerol (Gro) in POPC; and cholesterol and BHT (whole molecule and headgroup only). All the electron density profiles are symmetric, indicating that the bilayers are at equilibrium. As shown previously in detail, 34 the electron density profile for the pure POPC bilayer compares well with experiment. The thickness of the bilayer DHH was derived from the simulations of all the systems and is listed in Table 2. Although there is no experimental data on equimolar POPC/cholesterol bilayer, the value of DHH calculated from the simulations of the POPC/cholesterol bilayer (&DHH ' = 4.23 nm) is consistent with measurements on some related systems. Specifically, studies of bilayers containing a variety of phosphatidylcholines similar in size and (un)saturation to POPC mixed with 30–40 mol% cholesterol have DHH values around 3.9–4.8 nm. This amounts to an increase of about 10–25% with respect to the cor-

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responding pure phosphatidylcholine bilayer. 48–53 As &DHH ' = 3.50 nm in the pure POPC bilayer (Table 2), the thickening of the bilayer due to cholesterol (about 12%) is in line with experimental measurements. As observed in relation to the variation of AL in the presence of cholesterol and BHT, the electron density profile derived from the simulations of the POPC/BHT bilayer is intermediate between those for the pure POPC bilayer and the POPC/cholesterol bilayer. This is reflected in the bilayer thickness (&DHH ' = 3.88 nm) and the variations of the electron density across the bilayer. The latter shows properties shared with both a pure fluid bilayer and a liquid-ordered bilayer (as in a POPC/cholesterol bilayer). While cholesterol favours segregation between the leaflets as evidenced by the distinctive narrow dip in the total electron density profile in the centre of the membrane, the presence of BHT leads to a shallow minimum as in a pure POPC bilayer. Note, the plateau at |z| ≈ 1–1.5 nm and the shoulder at |z| ≈ 1 nm in Figure 5A in the total electron density profiles of the POPC/cholesterol and POPC/BHT bilayers respectively, are due to the steroid and hopanoid nuclei which reside in the phospholipid tail region. The peaks corresponding to the choline headgroup, phosphate and glycerol backbone in the POPC/BHT bilayer in Figure 5B lie between those for the POPC/cholesterol and POPC bilayers (closer to the POPC/cholesterol bilayer). They are narrower than the peaks for the POPC bilayer and of similar width to those for the POPC/cholesterol bilayer. This suggests that albeit lower than that of cholesterol, BHT still has a significant condensing effect on the lipid bilayer. As expected, the headgroup of cholesterol sits around the glycerol–ester region in POPC. 54–58 Because it is larger, the headgroup in BHT spans over the entire region at the lipid–water interface that consists of the glycerol backbone and the phosphocholine moiety.

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Ordering Effect of Bacteriohopanetetrol Tilt of the Lipid Molecules The distribution of the tilt angles γ1 and γ2 of the sn-1 palmitoyl and sn-2 oleoyl tails in POPC, respectively, in the simulations of POPC, POPC/cholesterol and POPC/BHT bilayers is shown in Figure 6, together with the distribution of the tilt angle γN of the main axis of the steroid (in cholesterol) and hopanoid (in BHT) nuclei in the simulations including cholesterol and BHT, respectively. Note, an angle of 0° corresponds to the axis parallel to the normal of the bilayer. On average, it was found that the distributions of γ1 and γ2 are comparable for a given system. The maximum values obtained in the pure POPC and mixed POPC/cholesterol and POPC/BHT bilayers are 21.5°, 9.5° and 12.5° for γ1 and 24.5°, 11.5° and 14.5° for γ2 , respectively. However, the shape of the distribution, while consistent across all three tilt angles, varies significantly between systems. The distributions of γ1 and γ2 are broad in the simulations of a POPC bilayer and extend up to about 100°. This indicates a high level of flexibility of the acyl chains. In the presence of BHT and cholesterol, the spread is dramatically reduced with the tail of the distributions stretching up to 50° and 40°, respectively, for both γ1 and γ2 . The width of the distributions between the two mixtures suggests that the orientational ordering effect of cholesterol is greater than that of BHT. The average orientation of cholesterol and BHT in the bilayer is similar with a maximum value of 9.5° for both molecules (Figure 6C). This is in good agreement with experiment for both cholesterol (γN around 11–19° 59–61 ) and BHT 62 that concluded that they lay almost perpendicular to the surface of the bilayer. The distribution of γN for BHT displays a longer right tail than that for cholesterol which implies that the orientation of BHT is less rigid than that cholesterol. Conformation of the Acyl Chains Ordering in the bilayer was characterised further by analysing the conformational disorder in the sn-1 palmitoyl tail of POPC. The fraction of gauche and trans rotamers in the tails is an important property in phospholipid bilayers and contributes to the quantification of the conformational disor-

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der in a lipid bilayer. The fraction of gauche defects was calculated in the simulations. It was found that, on average, the sn-1 pamlitoyl chain contained 3.1 gauche conformers in the pure POPC bilayer. Despite the lack of experimental measurements for a POPC bilayer, the value of ng compares favourably with other fluid, pure phosphatidylcholine bilayers: 2.7–2.85 for DLPC (1,2-lauroylsn-glycero-3-phosphocholine), 63,64 3 for DMPC 64 and 2.44–4.3 for DPPC. 63–69 The fraction of gauche rotamers was decreased by 10% (ng = 2.7) and almost 25% (ng = 2.3) in the bilayer containing BHT and cholesterol, respectively. To our knowledge, the only experimental reports of ng for cholesterol-containing bilayers are for liquid-orderered DPPC 70 and DMPC bilayers 64 containing about 30–33 mol% cholesterol where ng was estimated around 0.8–1.5. 64,67,70,71 Although, the value of ng is slightly higher in the POPC/cholesterol bilayer, it remains consistent with experiment. Figure 7 shows the probability distribution p(tn ) of sequences of n consecutive trans rotamers in the sn-1 palmitoyl tail in the POPC, POPC/cholesterol and POPC/BHT bilayers. Note, the sequence t13 corresponds to an all-trans conformation. As expected, p(tn ) decreases sharply as n increases in all systems. For all systems, p(t1) is around 0.4. In the POPC bilayer, the most common sequences involve one to four successive trans rotamers while the frequency of sequences t8 –t10 is negligible and that of t11 –t13 virtually zero. In presence of cholesterol and BHT, the number of short tn sequences (n ≤ 4) drops more quickly than in the POPC bilayer. Nonetheless, the decrease of p(tn) for sequences consisting of at least 5 trans bonds is slower and levels off at about 0.05 for t5–t8 in the presence of cholesterol. Interestingly, the variation of p(tn ) for the POPC/BHT bilayer is again intermediate between those of the pure POPC and POPC/cholesterol bilayers and overall closer to that of the POPC bilayer. In the POPC/cholesterol mixture, the probability of long trans sequences is markedly enhanced and never lower than 0.03, even for sequences t11 – t13 . This is in good agreement with infrared 67,70–73 and NMR 64,73–75 studies that suggested that the presence of cholesterol led to increased conformational order and a decrease in the number of gauche defects in acyl chains. In fact, cholesterol hinders the formation of gauche rotamers while promoting trans and all-trans conformers. 72

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Order Parameters of the Acyl Chains in POPC The extent to which covalent bonds within the lipids in a bilayer are ordered can be obtained experimentally using 2 H-NMR spectroscopy through the determination of a carbon–deuterium order parameter SCD . Relative differences in SCD values can be used to determine the state of the bilayer, e.g. lamellar (liquid-crystalline, liquid-ordered, gel), hexagonal and cubic. The SCD of the methylene and methanylylidene groups in the sn-1 palmitoyl and sn-2 oleoyl chains in POPC, was calculated for all the simulations. The |SCD | profiles for the POPC, POPC/cholesterol and POPC/BHT bilayers are displayed in Figure 8, together with experimental values determined by NMR spectroscopy for a pure POPC bilayer 76–80 and a 1:1 POPC/cholesterol bilayer. 80 Note the carbons in the acyl chains are numbered from the carbonyl group. Overall, the |SCD | values from all the simulations are in reasonable agreement with experiment. As expected, the sn-2 oleoyl tail displays the characteristic dip at positions 9 and 10 due to the double bond. The presence of cholesterol and BHT increases the |SCD | by almost a factor of 2–2.5 and 1.5–2, respectively. Again, the ordering effect of BHT is less than that of cholesterol. Although the ordering effect of cholesterol seems larger in the simulation than in experiment, 80,81 the average |SCD | calculated for the sn-1 palmitoyl chain in the simulations of POPC and POPC/cholesterol bilayers (0.19 and 0.35, respectively) is in line with the values reported by Nielsen et al. 82 (0.18 and 0.30 at 20°C in POPC and POPC + 30 mol% cholesterol bilayers, respectively), Kodati and Lafleur 73 (0.17 and 0.31 at 20°C in POPC and POPC + 30 mol% cholesterol bilayers, respectively) and Urbina et al. 83 (0.19 and 0.34 at 25°C in POPC and POPC + 30 mol% cholesterol bilayers, respectively). There is no equivalent experimental data for hopanoid-containing bilayers. However, the trend observed in the simulations of the POPC/BHT mixture is consistent with experiments that indicate that the addition of 20 mol% of aminobacteriohopanetriol to a DMPC bilayer leads to an increase in the first spectral moment (which is proportional to the average |SCD |) of the sn-2 myristoyl tail by about 50% at 30°C while the same amount of cholesterol augmented by 100%. 18

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Discussion For almost forty years, hopanoids have been known as ubiquitous molecules in rocks, sediments and crude oil, so much so they may be one of the most abundant biologically derived complex organic molecules on Earth. 84 These so-called geohopanoids originate from the abiotic degradation of hopanoids produced by bacteria (biohopanoids). Biohopanoids have been widely assumed to fulfil the function of sterols in bacterial membranes given their apparent chemical similarity. This view is based on pioneering studies in the 1980s–1990s that demonstrated that monolayers of phosphatidylcholine mixed with hopanoids underwent condensation 19–21 and that phosphatidylcholine vesicles tended to be more rigid 85 and less permeable 86 when a hopanoid was added. In all cases, the effect of hopanoids was analogous to that of cholesterol. It was also shown by NMR spectroscopy that, like cholesterol, aminobacteriohopanetriol increased order within the acyl tails in a DMPC vesicle. 18 Recently hopanoids have again been the focus of interest with the study by Sáenz et al. 17 showing that the simple C30 hopanoid diplopterol could induce the formation of a liquid-ordered phase (Lo ), similarly to cholesterol. However, as some bacteria such as Methylococcus species produce both sterols and hopanoids, these two classes of triterpenoids may not be entirely functionally equivalent. 87 The simulations in this work are aimed at furthering our understanding of the effect of hopanoids on biological membranes at a molecular level. In particular, we have attempted to answer the question of whether hopanoids and sterols were interchangeable as is generally assumed in the literature. The model systems investigated consisted of a pure POPC bilayer and a POPC bilayer mixed with either cholesterol, diploptene or bacteriohopanetetrol (BHT). Phosphatidylcholines are zwitterionic lipids and by far the preponderant class of phospholipids in the outer leaflet of mammalian plasma membranes. Although bacteria generally contain large amounts of either the zwitterionic phosphaditylethanolamines in Gram-negative bacteria or anionic lipids such as phosphatidylglycerols and cardiolipins in Gram-positive bacteria, 88 hopanoids were simulated in the presence of a phosphatidylcholine (that is POPC) in order to directly compare the effect of hopanoids with that of cholesterol in a well-characterised bilayer environment. Furthermore, as hopanoids have been 16


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identified in Gram-negative and Gram-positive bacteria, the nature of the headgroup may not have a preponderant effect on hopanoids–phospholipids interactions. The simulations show markedly different behaviour of two prototypical hopanoids—diploptene and bacteriohopanetetrol. When mixed with POPC in an equimolar ratio, BHT adopted an upright orientation similar to that of cholesterol. Specifically, the hopanoid nucleus aligned along the normal of the bilayer with the tetrol headgroup projecting towards the water. In contrast, diploptene migrated towards the core of the bilayer and lay in between the two leaflets (Figure 2). This occurred irrespective of the initial orientation of diploptene (headgroups facing outwards towards water or inwards towards the core of the bilayer) or the concentration (POPC–diploptene ratios of 1:1 and 10:1). Interestingly, the behaviour of diploptene is reminiscent of that of isoprene, 89 ubiquinones containing a long isoprenoid chain (6–10 isoprene units; as in coenzyme Q) 90,91 and squalane (the saturated form of squalene). 92 All these molecules have been shown to reside within the mid plane of a phospholipid bilayer and have been proposed to be synthesized by the cell in response to extreme environmental conditions. For example, isoprene is assumed to be involved in thermotolerance in plants. 89,93 Squalene in alkaliphilic bacteria 92 and long-chain ubiquinones (in particular coenzyme Q) in mitochondria in eukaryotes, 94 have been hypothesized to inhibit proton leakage across membranes. The behaviour of diploptene in the simulations is consistent with it having a kosmostropic role in response to certain environmental conditions. Diploptene has been found in the thylakoid membrane of cyanobacteria which is subject to a proton gradient (as in mitochondria). 95 Diploptene has also been identified in the ethanol-tolerant Zymomonas mobilis, and its content alongside that of other hopanoids is reportedly enriched when the bacterium is grown in high ethanol concentrations. 32 Remarkably, it has been shown that cholesterol displays a similar behaviour to diploptene in neutron diffraction experiments 96,97 and simulations 98 of lipid bilayers when mixed with polyunsaturated fatty acyl chains (PUFAs); namely it can migrate to the middle of the membrane. This striking deviation from the traditional upright orientation of cholesterol was explained by cholesterol having a preference for ordered regions, not highly disordered domains promoted by PUFAs. It is possible that the aggregation of diploptene between the two leaflets was

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partly due to the nature of the fatty acids in POPC. However, the palmitoyl and oleoyl tails used in the simulations are quite common in bacteria and this is not expected to play a major role. The simulations of BHT-containing bilayers showed that the behaviour of bacteriohopanetetrol is analogous to that of cholesterol in terms of its effect on the condensation and the enhancement of the ordering of phospholipids in a bilayer. This is in line with previous observations for BHT 19,21,85 and other hopanoids (diplopterol, 17 aminobacteriohopanetriol, 18 29a,29b-dihomohopan-29b-ol 86 and a 35-O-glucosyl BHT derivative 20). The properties calculated from the simulations of pure POPC and mixed POPC/cholesterol and POPC/BHT bilayers were in reasonable to very good agreement with the available experimental data, demonstrating the validity and the accuracy of the G ROMOS 54 A 7-force-field parameters used to describe cholesterol and BHT. The simulations allow a direct comparison of the effect of BHT and cholesterol on a range of structural properties known to be affected by the presence of cholesterol in eukaryotic membranes, including the area per lipid, the electron density profile across the bilayer, the tilt of the acyl chains and lipid molecules, the conformation of the sn-1 palmitoyl chain and the order parameters of the tails in a POPC bilayer. The condensing and ordering effect of cholesterol on lipid bilayers has been widely investigated by previous simulation studies, in particular by varying the concentration of cholesterol from almost 0 to 50 mol%. 99,100 Overall, BHT favoured ordering in the bilayer. However, the effect varied substantially with the property examined, highlighting the importance of considering a range of properties. The variations of the area per lipid AL (Figure 4 and Table 2) clearly show that the level of condensation of POPC in a lipid bilayer is of comparable magnitude in presence of BHT or cholesterol: the value of AL for POPC is 26% and 34% lower than in a fluid-phase POPC bilayer in POPC/BHT and POPC/cholesterol bilayers, respectively. The difference in the packing of POPC in the two mixtures may be partially due to the larger size of the headgroup in BHT than that in cholesterol. In contrast, the propensity of BHT to stabilise trans rotamers in the palmitoyl chains was on average significantly weaker than that of cholesterol. This is most evident in Figure 7 where the occurrence of 4 to 13 consecutive trans conformers along the palmitoyl chain remains almost constant at 0.03–0.05 in the simulations involving cholesterol whereas the

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occurrence of such long trans sequences decreases steadily with the length of the sequence to zero in the simulations of BHT, following closely the profile calculated for a pure POPC bilayer. Consequently, the presence of BHT has only a limited impact on the conformational dynamics of the acyl chains in the bilayer. The other properties investigated—the electron density profile across the bilayer, the tilt angles of the lipids and the order parameters of the tails—displayed intermediate features between a cholesterol-induced liquid-ordered bilayer and a liquid-crystalline bilayer. This means that the nature of the effect due to BHT and cholesterol are comparable but the magnitude of the change with respect to a fluid phase is greater with cholesterol than with BHT. It is especially evident in the variations of the electron density profiles in Figure 5 in which the separation of the peaks of individual components of POPC (choline, phosphate and glycerol) in the presence of BHT is almost halfway between that of pure POPC and cholesterol. On one hand, BHT and cholesterol restrict the mobility of the choline, phosphate and glycerol groups to a similar degree, but, on the other hand, the rigidifying and thickening effects of BHT on the bilayer is less than that of cholesterol. It is further illustrated in Figure 5A where the dip in the electron density profile calculated from the simulations containing BHT is of similar magnitude to that in the profile of pure POPC and not as deep as with cholesterol. This suggests that the ordering induced by BHT is not associated with an increased segregation of the the leaflets. The change in the distributions of the tilt angles γ1 and γ2 for the two tails, and γN for the steroid and hopanoid nuclei in cholesterol and BHT, respectively (Figure 6), together with the variations of the order parameter |SCD | in the sn-1 palmitoyl and sn-2 oleoyl chains in POPC (Figure 8) offer another way to characterise the relative effect of BHT and cholesterol. A previous simulation study 101 showed that these two properties are correlated in sterol-containing bilayers. It was found that the more ordered the bilayer, the lower the tilt angles of the tails and sterols. To some extent, this is also true in the presence of a BHT. Nonetheless, the comparison of the |SCD | profiles in Figure 8 reveals distinct ordering effect of the tails. Although cholesterol significantly enhanced ordering of the whole tails, its effect was most prominent in the region up to about carbons 8–10. It was less towards the methyl ends. In contrast, the relative increase of ordering due to BHT com-

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pared to the pure POPC bilayer was lower than that of cholesterol and primarily in the second half of the tails towards the terminal methyls. This is consistent with the density profile of cholesterol and BHT in Figure 5B (bottom panel) which shows that the electron density due to the hopanoid nucleus is maximum deeper in the bilayer than closer to the glycerol backbone. It also accounts for the difference observed between the distributions of the area per lipid AL of POPC in Figure 4 depending on whether AL was calculated using the position of the centre of mass of the headgroup or the position of the centre of mass of the whole lipid. Specifically, that the average value of AL for POPC in the POPC/BHT bilayer is greater when using the centre of mass of the headgroup indicates that the headgroups are more loosely packed than the whole lipids in presence of BHT, which means that tight packing of the tails is enhanced by BHT. The absence of such a difference when POPC is mixed with cholesterol is also a sign of the greater ordering effect of cholesterol which causes the POPC molecules to behave more like rigid rods. This is possibly due to the combined effect of the size of the headgroup and the fused-ring system in BHT and cholesterol. The small headgroup in cholesterol (a hydroxyl group) associated with a moderately bulky hydrocarbon chain in the tail region promotes condensation of POPC whereas the larger headgroup in BHT (5,6,7,8tetrahydroxy-sec-octyl group) and the five fused rings tend to have opposite packing-enhancing propensities. In summary, the precise location and the nature of the hopanoid and steroid nuclei in the bilayer leaflets directly impacts the ordering of the surrounding POPC molecules.

Conclusion Hopanoids have long been considered as bacterial surrogates for sterols despite there being few studies of the properties of hopanoid-containing membranes and little data at molecular or atomic level. In particular, the suggestion that they are involved in so-called “bacterial rafts” is primarily based on their apparent chemical similarity with sterols. 29–31 In this work, we have systematically compared the structural properties of two common hopanoids—bacteriohopanetetrol (BHT) and diploptene—with those of cholesterol in a raft-like lipid bilayer consisting of an equimolar

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mixture of POPC and cholesterol, BHT or diploptene. The effect of cholesterol and hopanoids on the lipid bilayer was also analysed with respect to a fluid, pure POPC bilayer. We have shown that diploptene and BHT exhibit different behaviours in the conditions of the simulations. Whereas BHT adopted a stable upright, cholesterol-like orientation in the lipid bilayer, diplotene partitioned in the mid plane of the bilayer, between the two leaflets, even at a lower concentration in the bilayer (9 mol%). The aggregation of diploptene between the two leaflets is consistent with diplotene enhancing the stability of membranes against chaotropic environmental conditions such as a proton gradient 95 and high ethanol concentrations. 32 In contrast, BHT acts more like cholesterol. However, the simulations clearly indicate that the ordering and condensing effect of BHT on a lipid bilayer is weaker than that of cholesterol. The detailed comparison of the effect of cholesterol and BHT on a lipid bilayer highlights that the contraction of a bilayer, its thickening and the ordering of the tails are in essence, intimately related but not consubstantial properties. Clearly, the chemical diversity of hopanoids may lead to a range of distinct properties that further simulation studies would contribute to examine in detail. Unlike sterols in eukaryotic membranes, hopanoids are likely to fulfil a broad range of functions in bacterial membranes, including promoting order in membranes in a cholesterol-like fashion (BHT) and stabilising membranes against environmental conditions as kosmotropic agents (diplotene).

Acknowledgement This work was funded from the National Health and Medical Research Council (NHMRC project grant APP1044327) with the assistance of high-performance computing resources provided at the National Computational Infrastructure National Facility systems at the Australian National University and iVEC at iVEC@Murdoch through the National Computational Merit Allocation Scheme supported by the Australian Government (project m39). The authors thank F. Separovic for helpful discussions and T. Mendes Ferreira for providing the experimental NMR data on carbon–hydrogen order parameters.

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Tables Table 1: Overview of the Systems Simulateda bilayer POPC POPC/cholesterol POPC/BHT POPC/diploptene

a b

c

number of POPCs 128 288 288 288 288 524 524

number of cholesterols 0 288 0 0 0 0 0

number of hopanoids 0 0 288 288 288 52 52

nature of the hopanoid — — BHT diploptene diploptene diploptene diploptene

number of waters 5941 15455 15455 15455 15455 15455 15455

initial orientation total of the headgroupb time (ns) outwards 400 outwards 450 outwards 450 c inwards 80–300 outwards 80–300 c inwards 80 outwards 80

BHT, bacteriohopanetetrol ((32R,33S,34S)-bacteriohopane-32,33,34,35-tetrol); POPC, 2-oleoyl-1-palmitoyl-snglycero-3-phosphocholine. The initial orientation of the lipid is either with the polar headgroup (choline in POPC, hydroxyl in cholesterol, 5,6,7,8tetrahydroxyoctan-2-yl in bacteriohopanetetrol and isopropenyl in diploptene) facing outwards towards water or inwards towards the centre of the bilayer. The headgroup of POPC faces outwards towards water while the headgroup of diploptene faces inwards towards the centre of the bilayer.

34


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Table 2: Structural Properties of the Bilayers Investigateda bilayer POPC POPC/cholesterol POPC/BHT a

(nm2 )

POPC 0.626 (0.001) 0.626 (0.001) 0.414 (0.002) 0.418 (0.002) 0.473 (0.002) 0.459 (0.002)

AL (nm2 ) cholesterol (nm2 ) — — 0.412 (0.001) 0.408 (0.002) — —

BHT (nm2 ) — — — — 0.477 (0.003) 0.492 (0.001)

DHH (nm) 3.50 (0.03) 4.23 (0.02) 3.88 (0.03)

AL , area per lipid; DHH , bilayer thickness. The values of AL calculated using the position of the centre of mass of the headgroup and whole molecule are in normal text and italics, respectively. The numbers between brackets are the standard deviations of the averages.

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Figure Captions Figure 1 Chemical structure and atom numbering of (A) cholesterol, (B) diploptene and (C) bacteriohopanetetrol. Figure 2 Initial and final structures of the simulations of hopanoid-containing bilayers. (A) POPC/diploptene bilayer with the diploptene initially set in an “inward” orientation. (B) POPC/diploptene bilayer with the diploptene initially set in an “outward” orientation. (C) POPC/BHT bilayer. The phosphorus atom and the tails in POPC are depicted as a yellow sphere and grey sticks, respectively. The atoms in the headgroup of diploptene and BHT are shown as light green and pink spheres while the hopanoid nucleus is in green and purple, respectively. Figure 3 Average orientation of diplotene in the simulations of equimolar POPC/diploptene bilayers were diploptene was initially set in an “inward” or “outward” orientation. (A) Time evolution of the average tilt angle &γD ' between the bilayer normal pointing away from the middle of the bilayer to bulk water and the main axis of the hopanoid nucleus in diploptene. Inset: Average tilt angle &γB ' between the bilayer normal pointing away from the middle of the bilayer to bulk water and the main axis of the hopanoid nucleus in bacteriohopanetetrol in the simulation of a POPC/BHT bilayer. (B) Time evolution of the average distance &zD ' between the centre of mass of the diploptene molecules in the upper and lower leaflets and the average phosphorus plane in the corresponding leaflet of POPC/diplopene bilayers. The distance is calculated along the bilayer normal (z-axis).

36


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Figure 4 Average distribution of the area per lipid AL for each type of lipid in the simulations. (A) Average distribution of AL for POPC in the POPC, POPC/cholesterol and POPC/BHT bilayers. (B) Average distribution of AL for cholesterol and bacteriohopanetetrol in the POPC/cholesterol and POPC/BHT bilayers, respectively. In each case, AL was calculated using the lateral position of either the centre of mass of the whole lipid molecules (denoted as “whole”) or the centre of mass of the headgroup only (“head”), that is phosphocholine in POPC and the non-cyclic moiety in cholesterol and bacteriohopanetetrol. Figure 5 Electron density profiles of the hydrated POPC, POPC/cholesterol and POPC/bacteriohopanetetrol bilayers. (A) Total electron density profiles. (B) Contribution of the individual components of the bilayer: choline (Cho), phosphate (P) and glycerol (Gro) of POPC, and whole molecule (total) and headgroup (head) of cholesterol and bacteriohopanetetrol. Figure 6 Tilt angle of the lipids in the POPC, POPC/cholesterol and POPC/bacteriohopanetetrol bilayers. (A–B) Average distribution of the tilt angle γ of the (A) sn-1 palmitoyl (γ1 ) and (B) sn-2 oleoyl (γ2 ) tails in POPC in the pure POPC, POPC/cholesterol and POPC/BHT bilayers. (C) Average distribution of the tilt angle γN of the steroid nucleus in cholesterol and the hopanoid nucleus in bacteriohopaneterol in the POPC/cholesterol and POPC/BHT bilayers, respectively. Figure 7 Occurrence of sequences tn (n = 1–13) of consecutive trans bonds in the sn-1 palmitoyl chain in the POPC, POPC/cholesterol and POPC/BHT bilayers.

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Figure 8 Deuterium order parameter |SCD | profiles of the sn-1 palmitoyl (Pam) and sn-2 oleoyl (Ole) acyl chains as a function of the carbon position calculated in the POPC/cholesterol and POPC/BHT bilayers. The |SCD | values are averaged over all the lipids and all the simulations of each system. Experimental |SCD | values: for sn-1 Pam, |SCD | measured by Ferreira et al. at 0% ( ) and 50% (

) cholesterol, 80 and Seelig and Seelig at 300 K 76 (

); for sn-2 Ole, |SCD | measured by Ferreira

et al. at 0% ( ) and 50% ( ) cholesterol, 80 Seelig and Waespe-Šarˇcevi`c 77 ( (

).

38


) and Perly et al. 79

Page 39 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21

22

27

25

A

20

18

H

17 11 1

H

19 9 10

H

3

8

26

13 14

H

5

HO

19

B 11 1

13

26

25 10

4

28

14

9 8

29

21

18

22

17

30

27

5

23

24

OH OH

19

C

18 11 1

25 10

4 23

13

26

14

9 8

29

21

28

17

OH 31

22 30

35

OH

27

5 24

Figure 1

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

POPC/diploptene (“inward” orientation)

B

POPC/diploptene (“outward” orientation)

C

Page 40 of 48

POPC/BHT

t = 0 ns

t = 0 ns

t = 0 ns

t = 300 ns

t = 300 ns

t = 450 ns

Figure 2

40


Page 41 of 48

A

60 50 20

30

〈γB〉 (º)

〈γD〉 (º)

40

20

10 0

10

0

100 200 300 400 time (ns)

0 0

B

50

100 150 200 time (ns)

250

300

250

300

1.0 0.5

〈zD〉 (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


upper leaflet

0.0 lower leaflet –0.5 –1.0 0

50

100 150 200 time (ns) inward outward

Figure 3

41



A

0.20

whole head

POPC

p(AL)

0.15 0.10 0.05

0.00 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 AL (nm2)

B

0.20

Cholesterol/BHT

0.15 p(AL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

whole head

0.10 0.05 0.00 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 AL (nm2) POPC POPC/cholesterol POPC/BHT

Figure 4

42


Page 42 of 48

A electron density (e∙nm–3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


500 400 300 200 100 0 –4 –3 –2 –1

0 1 z (nm)

B

2

3

4

3

4

Cho

100 electron density (e∙nm–3)

Page 43 of 48

P

100

Gro

100

total head

200 100 0 –4 –3 –2 –1

0 1 z (nm)

2

POPC POPC/cholesterol POPC/BHT

Figure 5

43



A 0.08


p(γ1)

0.06 0.04 0.02 0.00 0

20

40

B 0.08

p(γ2)

60 γ1 (º)

80

100

120


0.06 0.04 0.02 0.00 0

20

40

C 0.08

60 γ2 (º)

80

100

120

POPC/cholesterol POPC/BHT

0.06 p(γN)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.04 0.02 0.00 0

20

40

60 80 γN (º)

100

Figure 6

44


120

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0.5 0.4 p(tn)

Page 45 of 48


0.3 0.2 0.1 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 n

Figure 7

45



sn-1 Pam

0.4

sn-2 Ole

0.3 |SCD|

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.2 0.1 0.0 2

4

6

8

10

12

14 2 4 6 carbon atom positions

8

10

12

Figure 8

46


14

16


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Table of Contents graphic

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50x50mm (300 x 300 DPI)


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The Relative Effect of Sterols and Hopanoids on Lipid Bilayers: When

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